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BACTERIOLOGICAL REVIEWs, Sept. 1975, p. 186-196 Copyright © 1975 American Society for Microbiology Vol. 39, No. 3 Printed in USA. Crown Gall Tumors: Are Bacterial Nucleic Acids Involved? KARL A. DRLICAI* AND CLARENCE I. KADO Department of Plant Pathology, University of California, Davis, California 95616 INTRODUCTION ................................. ............................ 186 BACTERIAL NUCLEIC ACIDS AND TUMORIGENESIS ....................... 186 BACTERIAL NUCLEIC ACIDS AND ANTIGENS IN TUMOR CELLS ..... .... 187 Bacterial DNA ................................................................ 188 Bacterial RNA ................................................................ 190 Bacterial Antigens ............................................................ 191 BACTERIOPHAGES AND TUMORIGENESIS ......... ......................... 192 BACTERIAL PLASMIDS AND TUMORIGENESIS ........ ..................... 192 CONCLUDING REMARKS . ..................................................... 193 LITERATURE CITED . .......................................................... 193 INTRODUCTION Crown gall is a neoplastic disease of many dicotyledonous plants initiated during infection by Agrobacterium tumefaciens (Smith and Town) Conn. Transformation occurs within a few days after infection after which the pres- ence of living bacteria is no longer necessary to maintain the tumorous state (3, 16, 98). Bacte- ria-free tumor cells can be isolated and cultured indefinitely on chemically defined media lack- ing the phytohormones normally necessary for growth of plant cells in culture (14). Like many cultured animal tumor cell lines, cultured crown gall cells will proliferate as tumors when grafted onto suitable hosts (13) and in some cases pass on the transformed characters to host cells (58). During the last decade, preliminary findings from several laboratories indicated that bacte- rial nucleic acids could induce transformation (5, 6, 43, 90) and that tumor cells contained detectable amounts of nucleic acid (61, 65, 69, 72, 73, 77, 78, 81, 82) and antigens (20, 23, 24, 72, 76) presumably of bacterial origin. In every case the findings were equivocal, but collec- tively they suggested that bacterial nucleic acids might play a direct role in tumorigenesis. However, the results of subsequent control ex- periments now indicate that very little, if any, bacterial nucleic acid persists in crown gall tu- mor cells and that these tumor cells probably do not contain a full genome equivalent ofA. tume- faciens DNA. Bacteriophage PS8 of A. tumefa- ciens was also thought to be linked to crown gall tumorigenesis (64, 92, 93), but recent exper- iments cast doubt on the validity of this hypoth- esis as well. The most recent hypothesis main- tains that bacterial plasmids are involved in 1 Present address: Department of Biochemical Sciences, Princeton University, Princeton, N.J. 08540. tumor formation. Plasmids have been found in tumorigenic strains of A. tumefaciens (101), and strains that lose their plasmid also lose tumorigenicity (94). In this review, we examine the experimental evidence bearing on the hypotheses that A. tumefaciens nucleic acids, phages, or plasmids are involved in plant tumorigenesis. Readers interested in physiological aspects of the crown gall disease may wish to consult reviews by Beardsley (3), Braun (15), Kupila-Ahvenniemi and Therman (48), Veldstra (96), and Wood (99). BACTERIAL NUCLEIC ACIDS AND TUMORIGENESIS In 1947 Braun (12) mentioned, along with several other possibilities, that the tumor-in- ducing principle could be "a chemical fraction of the bacterial cell that is capable of initiating, as in the case of the transforming substance of pneumococci, a specific alteration in the host cell with a resultant continued and abnormal development of these cells." Early tests of this hypothesis involved inoculating plants with cul- ture media in which A. tumefaciens had grown (46, 56, 57, 91) and implicated bacterial deoxyri- bonucleic acid (DNA) as the tumor-inducing principle. Tumors formed at more than 60% of the inoculation sites, and treatment of the ex- tract with pancreatic deoxyribonuclease in- hibited tumor formation (46). However, steril- ity tests on the extracts were inadequate, and the claim that A. tumefaciens DNA-induced tumors was later retracted (45). In more recent studies, the bacterial contami- nation problem has been reduced by inoculat- ing plants (6, 8, 43, 90) or callus cultures (47) with sterile nucleic acid preparations extracted from A. tumefaciens. In the most convincing study (6), tissues proliferating at the inocula- 186 on June 2, 2020 by guest http://mmbr.asm.org/ Downloaded from

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Page 1: Crown Gall Tumors: Are Bacterial Nucleic Involved?gall disease may wish to consult reviews by Beardsley (3), Braun (15), Kupila-Ahvenniemi and Therman (48), Veldstra (96), and Wood

BACTERIOLOGICAL REVIEWs, Sept. 1975, p. 186-196Copyright © 1975 American Society for Microbiology

Vol. 39, No. 3Printed in USA.

Crown Gall Tumors: Are Bacterial Nucleic Acids Involved?KARL A. DRLICAI* AND CLARENCE I. KADO

Department ofPlant Pathology, University of California, Davis, California 95616

INTRODUCTION ................................. ............................ 186BACTERIAL NUCLEIC ACIDS AND TUMORIGENESIS ....................... 186BACTERIAL NUCLEIC ACIDS AND ANTIGENS IN TUMOR CELLS ..... .... 187

Bacterial DNA ................................................................ 188Bacterial RNA ................................................................ 190Bacterial Antigens ............................................................ 191

BACTERIOPHAGES AND TUMORIGENESIS ......... ......................... 192BACTERIAL PLASMIDS AND TUMORIGENESIS ........ ..................... 192CONCLUDING REMARKS...................................................... 193LITERATURE CITED........................................................... 193

INTRODUCTIONCrown gall is a neoplastic disease of many

dicotyledonous plants initiated during infectionby Agrobacterium tumefaciens (Smith andTown) Conn. Transformation occurs within afew days after infection after which the pres-ence of living bacteria is no longer necessary tomaintain the tumorous state (3, 16, 98). Bacte-ria-free tumor cells can be isolated and culturedindefinitely on chemically defined media lack-ing the phytohormones normally necessary forgrowth of plant cells in culture (14). Like manycultured animal tumor cell lines, culturedcrown gall cells will proliferate as tumors whengrafted onto suitable hosts (13) and in somecases pass on the transformed characters tohost cells (58).During the last decade, preliminary findings

from several laboratories indicated that bacte-rial nucleic acids could induce transformation(5, 6, 43, 90) and that tumor cells containeddetectable amounts of nucleic acid (61, 65, 69,72, 73, 77, 78, 81, 82) and antigens (20, 23, 24, 72,76) presumably of bacterial origin. In everycase the findings were equivocal, but collec-tively they suggested that bacterial nucleicacids might play a direct role in tumorigenesis.However, the results of subsequent control ex-periments now indicate that very little, if any,bacterial nucleic acid persists in crown gall tu-mor cells and that these tumor cells probably donot contain a full genome equivalent ofA. tume-faciens DNA. Bacteriophage PS8 ofA. tumefa-ciens was also thought to be linked to crowngall tumorigenesis (64, 92, 93), but recent exper-iments cast doubt on the validity ofthis hypoth-esis as well. The most recent hypothesis main-tains that bacterial plasmids are involved in

1 Present address: Department of Biochemical Sciences,Princeton University, Princeton, N.J. 08540.

tumor formation. Plasmids have been found intumorigenic strains of A. tumefaciens (101),and strains that lose their plasmid also losetumorigenicity (94).

In this review, we examine the experimentalevidence bearing on the hypotheses that A.tumefaciens nucleic acids, phages, or plasmidsare involved in plant tumorigenesis. Readersinterested in physiological aspects of the crowngall disease may wish to consult reviews byBeardsley (3), Braun (15), Kupila-Ahvenniemiand Therman (48), Veldstra (96), and Wood(99).

BACTERIAL NUCLEIC ACIDS ANDTUMORIGENESIS

In 1947 Braun (12) mentioned, along withseveral other possibilities, that the tumor-in-ducing principle could be "a chemical fraction ofthe bacterial cell that is capable of initiating, asin the case of the transforming substance ofpneumococci, a specific alteration in the hostcell with a resultant continued and abnormaldevelopment of these cells." Early tests of thishypothesis involved inoculating plants with cul-ture media in which A. tumefaciens had grown(46, 56, 57, 91) and implicated bacterial deoxyri-bonucleic acid (DNA) as the tumor-inducingprinciple. Tumors formed at more than 60% ofthe inoculation sites, and treatment of the ex-tract with pancreatic deoxyribonuclease in-hibited tumor formation (46). However, steril-ity tests on the extracts were inadequate, andthe claim that A. tumefaciens DNA-inducedtumors was later retracted (45).

In more recent studies, the bacterial contami-nation problem has been reduced by inoculat-ing plants (6, 8, 43, 90) or callus cultures (47)with sterile nucleic acid preparations extractedfrom A. tumefaciens. In the most convincingstudy (6), tissues proliferating at the inocula-

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tion site grew in vitro on media lacking cytoki-nins and auxins, factors normally required forin vitro growth of nontransformed plant cells.These cultured cells then proliferated into tu-mor-like tissues when grafted onto plants. How-ever, nucleases were not utilized to verify thata nucleic acid was indeed the inducing agent.Two similar reports which did not include nu-clease studies failed to agree upon whether bac-terial DNA or ribonucleic acid (RNA)-inducedtumors (43, 90). Neither of these studies ade-quately demonstrated formation offully autono-mous tumors; further study was needed to showthat the nucleic acid induced-tumors were capa-ble of growth in axenic culture in the absence ofphytohormones and that such cultured tissuescould proliferate as autonomous tumors whengrafted onto healthy plants. The most recentstudy of this type (5) involved inoculating A.tumefaciens RNA into Datura stramoniumstems which had previously been cut andplaced on an agar medium. Both distilled waterand RNA induced callus growth at the inocula-tion site, but only the RNA-induced outgrowthscontinued to proliferate when grafted onto freshplants. Although this was taken as evidence fortumor induction, no evidence was presentedthat the tissues had acquired the ability to growon agar media lacking cytokinins and phytohor-mones. The interpretation of this experiment isfurther complicated by the observation thatRNA extracted from either tumorigenic or non-tumorigenic bacteria was capable of producingthe effect (5).Although this direct approach has produced

promising preliminary findings, the resultshave been difficult to reproduce and often con-flict. One ofthe major problems is the lack ofanunequivocal assay for transformation. Forma-tion of a tumor-like outgrowth on a plant stemis insufficient evidence for tumor induction;some plants, such asDatura stramonium, read-ily form outgrowths when inoculated with non-tumorigenic bacteria (1, 37), tissue extracts (1),or even salt solutions such as ammonium chlo-ride (79). Tissue growth on minimal agar mediais also equivocal since normal tissues occasion-ally grow on minimal media (10, 28, 29, 53) andeven proliferate into tumor-like outgrowthswhen grafted onto plants (53).

Nucleases have been added to bacterial inoc-ula in attempts to demonstrate a role for nu-cleic acids in transforifation. It was found thathigh concentrations of pancreatic ribonucleaseA (2 to 4 mg/ml), but not deoxyribonuclease,inhibited bacterial initiation of tumors withoutaffecting the viability ofA. tumefaciens in cul-ture (17). This may indicate that RNA func-tions in tumorigenesis by carrying genetic infor-

mation. However, it is equally likely that ribo-nuclease affects transformation ofplant cells byaltering other aspects of bacterial metabolism;for example, the enzyme was shown to increasethe permeability ofA. tumefaciens cells to nu-cleotides (62). &

An effort has also been made to measure thetransfer of DNA from A. tumefaciens to plantcells (100). Radioactively labeled bacteria wereapplied to carrot root disks which were thenincubated, washed free ofbacteria, and assayedfor residual radioactivity. The bacteriawere labeled with [3H]thymidine, [ISI04,[14C]leucine or ["4C]uridine, and it was foundthat a greater percentage of radioactivity wasrecovered from carrot disks treated with[3H]thymidine-labeled bacteria than from bacte-ria labeled with uridine, sulfate, or leucine.The transfer reaction appeared to be specific toAgrobacterium since similarly treated Esche-richia coli cells transferred comparatively littletritium. The transfer apparently did not resultfrom generalized bacterial lysis since phage-induced bacterial lysis failed to increase trans-fer of the tritium label, and neither deoxyribo-nuclease treatment nor addition of nonradioac-tive thymidine reduced the percentage of trit-ium transferred. However, no evidence was pre-sented that the tritium transfer was directlyrelated to tumor formation, and since the triti-ated molecules transferred to the plant tissuewere not shown to be bacterial DNA, we cannotconclude that transfer of DNA was observed.Furthermore, accurate estimates ofthe quench-ing of each radioactive isotope by the carrotdisks are difficult to obtain; it is possible thatthe differences between the recovery of the dif-ferent isotopes reflect errors in the determina-tion of quenching rather than a preferentialtransfer of the tritiated molecules. The relativetransfer from [14C]- and [3Hlthymidine-labeledbacteria was not compared to test this possibil-ity.

Efforts to induce tumors with extracted A.tumefaciens nucleic acids, to inhibit tumor for-mation with nucleases, and to demonstratetransfer of DNA from A. tumefaciens to plantcells have collectively produced data consistentwith the concept that bacterial nucleic acidsplay a role in plant tumorigenesis. However,none of the evidence is conclusive and eachreport-must still be considered as a preliminaryobservation.

BACTERIAL NUCLEIC ACIDS ANDANTIGENS IN TUMOR CELLS

Two lines of evidence suggest that bacterialgenes influence tumor phenotypes. The first

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188 DRLICA AND KADO

observation was that crown gall tumors vary agreat deal in their rate of growth, degree ofautonomy, and state of differentiation, depend-ing upon the particular bacterial strains usedin transformation. For instance, bacterialstrain T-37 induces teratomatous tumors in to-bacco while strain B6 always causes fully auton-omous, fast-growing tumors (13). The secondobservation was that crown gall tumor tissues,in contrast to normal tissues, frequently con-tain the unusual amino acid derivatives octo-pine (59, 60), lysopine (9, 55), or nopaline (31),and that the presence or absence of the aminoacid derivative apparently depends upon thebacterial strain used to induce the tumor (30,67). In addition, many strains ofA. tumefaciensspecifically catabolize the same amino acid de-rivative (octopine or nopaline) found in crowngall tumors induced by that particular bacterialstrain (67). Furthermore, the ability to catabol-ize octopine or nopaline frequently parallels thecapacity of the bacterial strain to induce tumors(54, 68). The mechanistic relationship betweenthe production of a specific amino acid deriva-tive by a tumor and the ability of certain bacte-rial strains to induce tumors and to degrade thederivative is still unknown. The discovery ofthe unusual amino acid derivatives in normal(41, 80) and habituated (97) tissues (normal tis-sues capable of in vitro growth in the absence ofphytohormones) was thought to contradict thegeneralization that the presence of the deriva-tives in tumors was related to the bacterialstrain used to induce the tumor. These findingshave been reinvestigated (11, 52, 78), and it nowappears that other guanidine compounds in thenormal tissues had been misidentified as genu-ine octopine or lysopine. Bomhoff (11) showedthat the Nicotiana tabacum cv. Xanthi tissuesstudied by Johnson et al. (41) and thought tocontain octopine actually contain two interfer-ring compounds that are difficult to separatefrom octopine. Bomhoff (11) did confirm thatthe habituated tissue line from N. tabacum cv.White Burley examined initially by Wendt-Gal-litelli and Dobrigkeit (97) contained octopine.However, this particular tissue line had been inculture for more than two decades, and doubtsarose about its identity; consequently, Bomhoffexamined fresh habituated cultures derivedfrom N. tabacum cv. White Burley and Scorso-nera hispanica. No octopine was detected in thefresh cultures.

So far, the relatively few genetic experimentshave not indicated whether phenotypic differ-ences found in tumors arise from the continu-ous production of bacterial gene products bytumor cells or from a self-perpetuating induc-tion of plant genes by the bacterium at the time

of transformation. Since the first alternativepredicts that crown gall tumors should containbacterial nucleic acids and gene products, sev-eral laboratories have conducted searches forthese components in bacteria-free tumor cellcultures.

Bacterial DNA

Nucleic acid hybridization has been the prin-ciple technique employed in attempts to detectA. tumefaciens DNA in crown gall tissues. Forthe following discussion we have divided theexperiments into three categories based on theparameters measured. These parameters canbe summarized as measurement of (i) the frac-tion of radioactive, complementary RNA (syn-thesized in vitro from A. tumefaciens DNA)which could anneal with high concentrations oftumor DNA, (ii) the fraction of tumor DNAimmobilized on nitrocellulose filters whichcould be hybridized with radioactive A. tumefa-ciens DNA (or complementary RNA), and (iii)the acceleration of the rate of reassociation ofradioactive A. tumefaciens DNA by high con-centrations of tumor DNA.

In the first approach, radioactive RNA wasprepared in vitro using A. tumefaciens DNA asthe template and E. coli RNA polymerase asthe transcribing enzyme. This RNA was thenhybridized to an excess of denatured tumorDNA in solution. When increasing the tumorDNA concentration no longer increases the per-centage ofRNA hybridized, the hybridized frac-tion of the RNA then represents the percentageof the bacterial genome present in the tumorcells if the complementary RNA is a uniformtranscript. Normal callus DNA serves as a con-trol for nonspecific background and for hybridi-zation due to random sequence homologies. Wehave calculated from the data of Schilperoortand co-workers (72, 73, 78) that about 8% of thecomplementary RNA from A. tumefaciensDNA formed hybrids with tumor DNA whileonly 0.6% bound to normal DNA. However, twoimportant control experiments were omitted;first, no data were presented on whether thecomplementary RNA represented a uniformtranscript of the entire genome, making anyquantitative conclusion questionable. Sec-ondly, the base-pairing fidelity of the duplexesscored as hybrids was not analyzed; conse-quently, we cannot be certain that bonafideRNA-DNA hybrids were observed.The second approach, called DNA saturation

hybridization, involved annealing denatured,radioactive A. tumefaciens DNA to denaturedtumor or normal DNA immobilized on nitrocel-lulose filters. At saturating concentrations of

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radioactive DNA, the ratio of the radioactivebacterial DNA annealed to the filter-bound non-radioactive tumor DNA estimates the fractionof the tumor DNA having sequences comple-mentary to the bacterial DNA and presumablyof bacterial origin. In actual practice, true satu-ration values cannot be obtained because therate of self-renaturation in solution is greaterthan the rate of annealing of the radioactiveDNA in solution with the nonradioactive DNAimmobilized on the filter. Consequently, experi-mental saturation values tend to underesti-mate the degree of base sequence homology andmust be calibrated with saturation values ob-tained from model filters containing small,known amounts of A. tumefaciens DNA mixedwith large amounts of normal plant DNA. Twopreliminary saturation-hybridization studies(69, 81) have often been cited as evidence forsequence homologies between A. tumefaciensand crown gall tumor DNA. However, the an-nealing systems were not calibrated with modelfilters nor were the thermal stabilities of theputative DNA-DNA duplexes examined. As aresult, these experiments cannot serve as evi-dence for the presence ofbase sequence homolo-gies. We have recently conducted the same typeof study (26) using more stringent reassociationconditions and found little, if any, tumor DNAhomologous to A. tumefaciens DNA. Similarwork by Farrand, Eden, Bendich, Gordon, andChilton (personal communication) also failed todetect bonafide bacterial/tumor DNA-DNA du-plexes.

In our studies (26) the hybridization systemswere calibrated with model filters containingsmall, known amounts ofA. tumefaciens DNAmixed with normal plant DNA. Using this cali-bration, we estimated that the maximum se-quence homology was 0.02%. As a point of refer-ence, 0.1% homology is equivalent to one tothree bacterial genomes per diploid plant cell.

Schilperoort et al. (77) modified the DNAsaturation approach by substituting radioac-tive RNA synthesized in vitro for radioactive A.tumefaciens DNA. They concluded that 0.9%o ofthe crown gall DNA hybridized with RNA syn-thesized from A. tumefaciens DNA and 1.8%with RNA complementary to A. tumefaciensbacteriophage PS8 DNA. However, these con-clusions were subject to the same types of criti-cisms that apply to the experiments describedabove. Quantitative interpretations could notbe made because the fidelity of base pairingwas low; the thermal dissociation midpoint ofhybrids formed with tumor DNA was 8 C lowerthan that of hybrids with bacterial or phageDNA and only 2 C higher than the thermalmidpoint of nonspecific hybrids formed with

normal plant DNA (95). In addition, no datawere presented to test whether the complemen-tary RNA represented a uniform transcript ofthe entire bacterial or phage genomes. More-over, efforts by another laboratory to confirmand extend these hybridization results havebeen unsuccessful (27). Subsequently, Schilpe-roort et al. (75) re-examined their original workand concluded that no hybrids had formed be-tween tumor DNA and complementary RNAfrom either A. tumefaciens or bacteriophagePS8 DNA.The third approach used to search for bacte-

rial DNA sequences involves measuring therate of reassociation of radioactive bacterialDNA in the presence of large amounts of nonra-dioactive normal or tumor DNA. BacterialDNA sequences present in the tumor DNA willincrease the apparent reassociation rate of thelabeled bacterial DNA. The technique is poten-tially very powerful; it is possible to determinehow much tumor DNA is homologous to bacte-rial DNA and whether the tumor DNA containsall of the bacterial nucleotide sequences. Thismethod has been used by two laboratories withconflicting results. Chilton et al. (21) detectedno increase in the reassociation rate ofA. tume-faciens DNA in the presence of crown gall tu-mor DNA. They assessed the sensitivity oftheir assay by conducting reconstruction experi-ments in which the reassociation rate ofradioac-tive A. tumefaciens DNA was measured in thepresence of small, known amounts of nonradio-active A. tumefaciens DNA mixed with salmonDNA. From these results, Chilton et al. (21)concluded that no more than 0.01% ofthe tumorDNA shared sequence homologies withA. tume-faciens DNA, an amount equal to 0.1 to 0.3genome equivalents of bacterial DNA per dip-loid plant cell.

In contrast, Patillon (65) reported that crowngall tumor DNA increased the reassociationrate of A. tumefaciens DNA by the amountexpected if the tumor DNA contained a fullgenome equivalent of bacterial DNA. Since thistumor DNA had been enriched for the uniquesequences in the plant genome, the sensitivityof Patillon's assay may have been 2 to 5 timesgreater than that of Chilton et al. (21). Such anincrease might explain the apparent discrep-ancy. However, Patillon's observations (65) arenot complete enough to be considered as strongevidence for DNA sequence homologies be-tween bacterial and tumor DNAs. First, nor-mal tissue DNA was not included as a controlfor nonspecific sequence homologies. Second,the annealing system was not properly cali-brated by measuring the reassociation rate ofradioactive A. tumefaciens DNA in the pres-

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190 DRLICA AND KADO

ence of small known amounts of nonradioactiveA. tumefaciens DNA and normal callus tissueDNA. Third, the base-pairing fidelity of themolecules scored as hybrids was not assessed bythermal dissociation studies.The nucleic acid hybridization studies have

not demonstrated the presence of bacterialDNA sequences in sterile crown gall tissues;none of the positive reports included thermalelution profiles showing a high degree of base-pairing fidelity. On the other hand, the tech-niques used in the negative reports were notsensitive enough to eliminate the possibilitythat tumor cells contain a small fraction of thebacterial genome. Moreover, none of the celllines were cloned from a single cell; conse-quently, normal cells might be present in tu-mor lines and require modification of the sensi-tivity estimates of the negative experiments.Some of the findings by Qu6tier et al. (69)

may explain why hybrids having a low base-pairing fidelity form between A. tumefaciensDNA and crown gall DNA. DNA extractedfrom soybean tissues, which had been woundedbut not transformed, contained a satellite DNArich in guanosine and cytosine. Plant ribosomalRNA (rRNA) hybridized with the satelliteDNA, suggesting that rRNA genes (rDNA)were amplified during the wound-healingprocess. A. tumefaciens DNA also hybridizedwith the satellite DNA. If the amplification ofrDNA is a general feature associated withwounding and persists during transformation,the hybrids between A. tumefaciens DNA andtumor DNA in other experiments (69, 81) mayactually be mispaired structures composed ofA.tumefaciens DNA and tumor rDNA. Theknown base sequence similarities between bac-terial and plant rRNAs (7) and the use of non-stringent annealing conditions make this aviable alternative explanation. Amplification ofrDNA during plant development has been re-ported (2), but the lack of rRNA-DNA hybridthermal stability data and the possibility ofbac-terial contamination in the plant material makeconclusions about amplification still tentative(38, 66).Cesium density-gradient analyses of plant

DNA preparations from sterile tissue culturessuggested that tumor DNA contains a satellitenot found in DNA preparations from normaltissues (32, 82). Srivastava and Chadha (82)reported that the satellite was rich in guano-sine and cytosine, and since A. tumefaciensDNA has about 50% more guanosine and cyto-sine than plant DNA, the authors proposedthat the satellite was actually bacterial DNA.The satellite was detected only after the tobaccotumor DNA was sheared, indicating that the

satellite was linked to plant DNA prior toshearing. However, this latter result could notbe duplicated in either Schilperoort's (73) or ourown laboratory. Moreover, DNA from sometumor cell lines lack the characteristic satelliteDNA, whereas it has been found in DNA fromboth plant seedlings and cultured habituatedcells (70). Satellite DNA also appears soon afterNicotiana glauca pith tissues are transferredfrom the plant to agar media (63), but severaldays later this satellite DNA disappears. It isnot known whether the various satellite DNAsare related; however, there is no good evidencethat any ofthe satellites contain base sequenceshomologous to those in A. tumefaciens DNA.

Bacterial RNACultured tumor tissues have been assayed for

A. tumefaciens RNA by RNA-DNA hybridiza-tion experiments. Tumor RNA, fractionated bysucrose density-gradient centrifugation, wasfound to hybridize to bacterial DNA 10 to 20times as much as RNA extracted from normaltissues (61). However, this annealing experi-ment was conducted under nonstringent condi-tions and the thermal stabilities of the hybridswere not examined. Consequently, a firm con-clusion cannot be drawn. Tumor messengerRNAs were examined by filter hybridization inour laboratory (42). The binding of these mes-senger RNAs to bacterial DNA filters was fourtimes greater than the binding of RNAs fromcomparable normal tissue, but thermal dissocia-tion analyses showed that this was due to non-specific binding (42). Hence, as in the case ofbacterial DNA, there is no strong evidence thattumor cells contain bacterial RNA.

Stroun et al. (88) concluded that there may bea direct relation between the appearance ofA.tumefaciens RNA in plant cells and tumor in-duction. Tomato plants which had first takenup living bacteria through the cut ends ofshoots later developed tumors at the sites ofsecondary wounds. However, no tumors formedif the plants were treated soon after woundingwith rifamycin, an antibiotic which in parallelexperiments inhibited bacterial but not plantRNA polymerases (84). Radioactive RNA ex-tracted from the plants was analyzed by molecu-lar hybridization to bacterial or plant DNAimmobilized on nitrocellulose filters. Plantstreated with bacteria contained RNA whichbound to bacterial DNA on filters, and thisRNA was not present in plants also treatedwith rifamycin. Several lines of evidence sug-gested to Stroun et al. (88) that the bacterialRNA they detected was synthesized insideplant cells rather than in living bacteria resid-ing in the plant. First, living bacteria were

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found only in the xylem tissues, and these bacte-ria incorporated very little of the radioactiveuridine used to label the RNA (85, 86) Second,surgical removal of the xylem and bacteriaprior to labeling had no effect on the RNADNAhybridization results in a parallel study withanother plant species (85, 87, 89). Third, treat-ment of the plant with the bacteriocides chlor-amphenicol or colimycin (85, 87) also had noeffect on the RNADNA hybridization results.

Stroun et al. (88) also reported that part ofthe radioactive DNA extracted from plantstreated with bacteria had a buoyant densityintermediate to the densities of bacterial andplant DNAs when analyzed by cesium chloridedensity-gradient centrifugation. Thermal dena-turation of the DNA of intermediate densityproduced only a single peak in cesium chloridegradients while extensive shearing of the DNAresolved it into two species, one at the densityofbacterial DNA and the other at the density ofplant DNA. Consequently, it is possible, asStroun and co-workers (88) suggest, that theDNA of intermediate density represents bacte-rial DNA fragments covalently joined to plantDNA and that this DNA may even be the tem-plate for the RNA species discussed above. How-ever, such a conclusion must be considered verytentative; buoyant density measurements arenot sufficient for the identification of bacterialDNA and the necessary nucleic acid annealingstudies have not yet been conducted.The relationship of Stroun's work to the

crown gall disease is ambiguous. It is difficultto rule out the possibility that the RNA speciesbeing detected were synthesized by contaminat-ing bacteria residing in the plant, and the rifa-mycin treatment may have simply killed theliving bacteria known to be necessary for trans-formation. Moreover, the findings from the nu-cleic acid analyses were similar whether or notthe plants received the wounding treatmentrequired for tumor formation (85, 87, 88). It iseven questionable that cells actually undergo-ing transformation represented a measurablefraction of the sample analyzed. Furthermore,the appearance of bacterial nucleic acids in theplants did not arise from the tumorigenic prop-erties ofA. tumefaciens; identical results wereobtained when either E. coli or Pseudomonasfluorescens were substituted for A. tumefaciens(83-87). Consequently, these studies do not sup-port the hypothesis that bacterial nucleic acidsplay a direct role in tumorigenesis.

Bacterial AntigensSeveral preliminary immunodiffusion and

complement-fixation studies have suggestedthat tumor tissues contain bacterial specific an-

CROWN GALL TUMORS 191

tigens. Antibodies formed against soluble anti-gens from A. tumefaciens cross-reacted withsoluble antigens extracted from sterile tumortissues but not with those from normal tissues(20, 23, 72, 76). Likewise, A. tumefaciens-solu-ble antigens cross-reacted with anti-tumor serabut not with sera formed against normal tissueantigens. When antitumor sera were first re-acted with normal tissue antigens and thenwith A. tumefaciens antigens, three (20) or four(72, 76) cross-reacting species are formed whichare destroyed by heat or Pronase treatment (72,76), indicating that these antigens may be pro-teins.Although these immunological results may

reflect the expression of bacterial genes in tu-mor cells, their biological significance is uncer-tain because A. tumefaciens, normal plantcells, and habituated plant cells (23, 24) appar-ently share cross-reacting substances. More-over, Bomhoff (11) has recently shown thatpreimmune sera as well as sera against normalplant tissues cross-react with soluble extractsfrom A. tumefaciens and both normal andcrown gall callus tissues. In no case have thecross-reacting substances been identified, so itis even uncertain that an antigen-antibody in-teraction was being observed. Consequently, itmust be concluded that the experimental evi-dence does not establish the presence of bacte-rial antigens in crown gall tumor cells.

Experiments with other plant systems indi-cate that plants do have the ability to expressbacterial and bacteriophage genes. Carlson (19)infected tobacco protoplasts with bacteriophageT3 and observed the appearance of two enzy-matic activities associated with T3 gene prod-ucts. Using the same rationale, Doy et al. (25)treated tomato and Arabidopsis callus cultureswith specialized transducing bacteriophages(180 and X) containing the genes of E. coligalactose (gal) and lactose (lac) operons. Theplant cells acquired the ability to grow on me-dia containing galactose or lactose as the bulkcarbon source. Phages carrying defective galac-tose genes, other bacterial genes, or no bacte-rial genes failed to convert the plant cells. Inthe case of conversion of plant cells to grow onmedia containing lactose as the sole source ofbulk carbon, the specific activity ofl3 galactosid-ase increased about 30-fold and a major portionof the enzyme activity was protected from heatinactivation by antisera formed against E. coli,3-galactosidase. (3-Galactosidase activity inplant cells not treated with the phage remainedheat sensitive after treatment with the anti-sera. In another experiment callus culturestreated with a phage carrying an E. coli mu-tant suppressor gene (Sup F+, which inserts

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192 DRLICA AND KADO

tyrosine at UAG nonsense codons) lost the abil-ity to grow on media normally optimal for plantcell growth. Using sycamore cells, Johnson etal. (40) reported findings similar to Doy et al.(25), but they were unable to detect ,&galactosid-ase either immunologically or by enzyme as-

say.

BACTERIOPHAGES ANDTUMORIGENESIS

A. tumefaciens bacteriophages have alsobeen considered as the carriers of the informa-tion responsible for transformation (3, 51, 92,93). Although purified phages have failed toinduce tumor formation (4), the reports claim-ing that phage DNA base sequences occur intumor cells (76) and that coliphages can trans-fer genes to plant cells (19, 25, 39, 40) lendsupport to the concept. The phage hypothesiswas based on the following observations: (i)

treatments which stimulate phage productionin lysogenized bacteria also increase the spe-

cific tumorigenicity of A. tumefaciens (33-36);(ii) extracts of cultured tumor tissues inducelysis in phage-sensitive strains of A. tumefa-ciens (64, 92); (iii) ultrastructural studies indi-cate that lysed bacteria and free phages arepresent in inoculated wounds (49, 50); and (iv)according to Leff and Beardsley (51), phageDNA induced tumors. However, specific tumori-genicity of the bacteria could also be increasedwithout phage production (33), plaque-formingactivity is often not found in tumors (77), induc-tion of tumors by phage DNA has not beensubstantiated by other laboratories (4), and a

bacterial strain cured of PS8 phage was stillfully tumorigenic when assayed for tumor-form-ing ability on pinto bean leaves (18). Since PS8phage DNA could still be present in cured bacte-ria and function in tumorigenesis, the curedbacteria were examined for residual phageDNA by nucleic acid hybridization techniques(22). Radioactive bacterial DNA was annealedto PS8 phage DNA immobilized on nitrocellu-lose filters and no hybrids were found. Aftercorrecting the data of DeLey et al. (22) for spe-cific radioactivity, we calculated that less than12% as much DNA from the cured bacterialstrain hybridized to PS8 phage DNA as didDNA extracted from the parental strain. Quan-titative estimates of the amount of PS8 phageDNA present in the bacterial DNA samplescannot be derived from the data because experi-ments with high ratios of filter-bound PS8phage DNA to bacterial DNA in solution were

not conducted. However, if the parent bacterialcell contained only one copy of the phage ge-nome, as has been shown by nucleic acid hybrid-ization for another lysogenized strain of A. tu-

mefaciens (77), then only 12% or one of eightcured bacteria could contain an intact phagegenome. Although the data were not presented(18), such decreases in the fraction of tumori-genic bacteria would have been detectable bythe pinto bean leaf tumor assay. Since only asmall fraction of the PS8 phage genome mightbe required for tumorigenesis, all of the curedbacterial cells could still contain enough phageDNA to retain their full tumorigenic capacity.Even sensitive hybridization analyses, such asthose of Schilperoort et al. (79) which show thatthe virulent A6 strain contains less than 1% ofthe PS8 phage genome, do not rule out a viraletiology for crown gall because a phage unre-lated to those tested may be responsible fortransformation.As mentioned above, it has been reported (77)

that sterile tumor cells contain PS8 phage DNAsequences. This report has recently been chal-lenged by three types of nucleic acid hybridiza-tion studies: (i) PS8 phage DNA/tumor DNAfilter saturation (Farrand et al., personalcommunication); (ii) PS8 complementaryRNA/tumor DNA filter saturation (27); and (iii)PS8 DNA/tumor DNA solution reassociation ki-netics (21). In each case, the hybridization sys-tem was calibrated by reacting radioactivephage nucleic acids with known amounts ofPS8DNA mixed with salmon DNA, and in no casewas phage DNA detected in tumor DNA. Themost sensitive method, renaturation kinetics(21), indicated that less than 0.0007 to 0.001% ofthe tumor DNA, i.e., less than one phage ge-nome equivalent, could be homologous to PS8DNA. There is still no universally acceptedevidence that PS8 phage and tumor DNAsshare homologous base sequences or thatphages play a direct role in tumorigenesis.

BACTERIAL PLASMIDS ANDTUMORIGENESIS

Recently it has been shown that plasmids arepresent in A. tumefaciens (101). These plasmidsare of relatively high molecular weight, rang-ing from 96 x 106 to 156 x 106 depending uponthe bacterial strain in which the plasmid wasdetected. So far, these large plasmids have beenfound in all tumorigenic bacterial strains exam-ined while they have been absent in most non-tumorigenic strains of the genus Agrobacte-rium. The correlation between tumorigenicityand the possession of a plasmid suggests thatplasmids may be involved in the transforma-tion process. Such a conclusion requires thatthe loss or gain of tumorigenicity by a bacterialstrain be directly associated with the loss orgain of the plasmid by the bacterial strain. Thisrequirement has been partially satisfied.

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Growth of A. tumefaciens strain C58 at 37 Ccures the strain of its plasmid at the same ratethe strain loses its tumorigenicity (94). It hasalso been found that A. tumefaciens strainssensitive to agrocin 84, a bacteriocin producedby A. radiobacter, were tumorigenic (44, 71)and contained a plasmid (71); strains resistantto agrocin 84 were nontumorigenic (44, 71) andno longer harbored a plasmid (71). As expected,A. tumefaciens C58 cured of its plasmid andrendered non-tumorigenic by growth at 37 Cwas also resistant to agrocin 84 (71). Tumori-genicity has not yet been conferred on a non-tumorigenic bacterial strain by the acquisitionof a plasmid.

CONCLUDING REMARKSThe hypotheses that A. tumefaciens nucleic

acids, phages, or plasmids are directly involvedin transformation of plant cells are consistentwith the experimental results. However, thereis no strong, unequivocal evidence favoring onehypothesis over another. Live bacteria are nec-essary for crown gall tumor initiation; no sys-tem has been developed in which isolated bacte-rial components reproducibly induce crown galltumors. Nucleic acid hybridization studies havebeen reported which claim to show homologiesbetween bacterial or phage DNA and tumornucleic acids; each has been subsequently chal-lenged. On the other hand, no experimentalevidence rules out the idea that a small part ofthe bacterial genome (or phage or plasmidDNA) invades the plant cell, although recentnucleic acid hybridization studies (21, 26, 27)argue against each tumor cell containing theentire bacterial genome. Indeed, the observa-tion that the properties of plant cells can bealtered in predictable ways by coliphagesmakes such a model quite feasible.More than a quarter century has passed since

the search for the chemical identity of the tu-mor-inducing principle was begun; it still hasnot been found. Perhaps the latest clue, thecorrelation between plasmid-containing Agro-bacterium strains and tumorigenicity, will leadto the answer.

ACKNOWLEDGMENTSWe thank F. Meins, R. A. Langley, T. Kosuge, J.

M. Gardner, N. Sueoka, S. Deeb, and D. M. Lin-strom for stimulating discussions and many helpfulsuggestions.

This work was supported by grant CA-11526 fromthe National Cancer Institute and by Cancer Re-search Funds from the University of California. K.A. Drlica was supported by American Cancer Soci-ety Dernham fellowship J-183.

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